Talk given at the 2018 Association of Biomolecular Resource Facilities Mid-Atlantic Directors and Staff of Scientific Cores meeting

1. Stephen D. Turner, Ph.D.
Bioinformatics Core Director
University of Virginia School of Medicine
bioinformatics.virginia.edu
@strnr
Tools for Improving Rigor &
Reproducibility in Bioinformatics
Slides: bit.ly/madssci2018repro

2. We Are in the Middle of a
New Movement in Genomics
• Genomics/bioinformatics advancing at grueling pace
- New questions
- New study designs
- New technologies, new [fill-in-the-blank]-seq
• New movements have:
- Leaders / method developers / early adopters
- First followers
- Everybody else
• New technology leads to more reproducibility issues
2
CORES!

3. Reproducibility is hard!
• Genomics data is too large and high
dimensional to easily inspect or visualize.
• Workflows involve multiple steps and it's hard
to inspect every step.
• Unlike in the wet lab, we don't always know
what to expect of our genomics data analysis.
• It can be hard to distinguish good from bad
results.
3

4. 4

5. Reproducibility:
What's in it for you?
• Your future self will thank you
- Re-running analysis with different parameters
- Re-running analysis with new data
- Documentation
• Faster/cheaper
- Modular workflows
- Reusable code chunks
• Makes collaboration with others easier
5
"Robust research is about doing small things that
stack the deck in your favor to prevent mistakes."
–Vince Buffalo, author of Bioinformatics Data Skills (2015).

6. Obstacles to Reproducibility
1. Bioinformatics software
2. Pipeline / workflow management
3. Documentation
4. Data / code sharing
6
A non-comprehensive list of

7. Bioinformatics
Software
7

8. Bioinformatics Software
• Bioinformatics software implements complex algorithms.
- Dozens of parameters, endless permutations
- Defaults not always optimal
• Perception:
8
ACACTCGCATCCGCACATCGCACTA
GGTCAGCATACGCCGACTCCGACCG
GCGCTATCGCCAGCGGAAATCGCAA

9. Bioinformatics Software
• Bioinformatics software implements complex algorithms.
- Dozens of parameters, endless permutations
- Defaults not always optimal
• Reality: Software is written by smart people, but:
- Not software engineers
- Not using good practice (version control,
modularization, commentary, testing)
- Unable to offer long-term
maintenance / support
- Focus on graduating /
publishing, not support
- Not always easily available
9

10. • Missing or incomplete documentation
• Distribution is missing files
• Missing third party package
• Dependencies failed to build
• Runtime error
• Internal compiler error
• My last week:
- samtools: error while loading shared libraries:
libbz2.so.1.0: cannot open shared object file
- error while loading shared libraries: libz.so.1:
failed to map segment from shared object:
Operation not permitted
- /lib64/libc.so.6: version `GLIBC_2.14' not found

11. 11
https://twitter.com/ianholmes/status/288689712636493824

12. Package managers
12
Mac OS WindowsLinux
apt-get
yum
homebrew
macports
?????
?????
Cross-platform

13. Conda
• Cross-platform package manager: Win, Mac, Linux
• Language agnostic (can be used to install C/C++,
Fortran, Go, R, Python, Perl, Java, etc.).
• User-installable – no admin/root privileges needed.
• Describes packages with a recipe defining
dependencies and a build script that installs.
• Channels: conda provides many common packages by
default. Additional channels add more.
• Isolated environments
- Versions and tools can be managed per-project
- No conflicts or version incompatibility
- Environments can be shared via simple text files
13

14. Conda: Main commands
• conda create -n <environment>
• source activate <environment>
• conda search <package>
• conda install <package>
• conda upgrade <package>
• conda uninstall <package>
14

15. Conda: example
• Create a new environment named madssci:
conda create -n madssci
• Activate that environment
source activate madssci
• Install some packages
conda install blast bioconductor-flowcore
• Install a particular version
conda install samtools=0.1.19
15

16. Bioconda
• bioconda.github.io
• Bioconda is a channel for the conda package manager
• Repository for more than 3,000 bioinformatics
packages ready to use with conda install
• >250 contributors have added/updated recipes
• Preprint: Grüning, Björn, et al. "Bioconda: A sustainable
and comprehensive software distribution for the life
sciences." bioRxiv (2017): 207092.
https://www.biorxiv.org/content/early/2017/10/27/207092
• See also: "Nature TechBlog: Bioconda Promises to Ease
Bioinformatics Software Installation Woes"
http://blogs.nature.com/naturejobs/2017/11/03/techblog-bioconda-
promises-to-ease-bioinformatics-software-installation-woes/
16

17. Docker
• docker.com
• Lightweight virtualization technology
• Package software with all of its dependencies into an isolated "container"
• Containers have everything needed to run: code, system tools & libraries
• Like VMs: portable. = reproducibility!
• Unlike VMs: containers virtualize the OS instead of the hardware. = More
efficient, more portable. Near native performance, instant startup, small
images. Easy to share.
• https://www.docker.com/what-container
• https://blog.docker.com/2016/03/containers-are-not-vms/
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Containers are an abstraction at
the app layer that packages code
and dependencies together.
Multiple containers can run on the
same machine and share the OS
kernel with other containers, each
running as isolated processes in
user space. Containers take up
less space than VMs (container
images are typically tens of MBs in
size), and start almost instantly.
Virtual machines (VMs) are an
abstraction of physical hardware
turning one server into many
servers. The hypervisor allows
multiple VMs to run on a single
machine. Each VM includes a full
copy of an operating system, one
or more apps, necessary binaries
and libraries - taking up tens of
GBs. VMs can also be slow to
boot.

18. Pipeline / workflow
management
18

19. Pipeline / Workflow Management
• Bioinformatics data analysis: series of steps
involving many different programs tied together
with file-based inputs and outputs. E.g.:
19

20. Pipeline / Workflow Management
• Simple solution: simple (bash) script
- List of commands
- Pros: quick, easy, portable, universal
- Cons: not scalable, no re-entry / partial execution,
assumes dependency availability, difficult / no
parallelization
• Workflow management systems
- Make (installed on most systems)
- Snakemake
- Nextflow
- Galaxy
- Many more: github.com/pditommaso/awesome-pipeline
20

21. Nextflow
• nextflow.io
• Di Tommaso, Paolo, et al. "Nextflow enables reproducible
computational workflows." Nature biotechnology 35.4
(2017): 316-319.
• Features:
- Free
- Actively developed
- Supports docker containers
- Easy parallelization, implicitly defined
- Continuous checkpointing & resumed execution
- Easily portable across architectures (SGE, LSF,
SLURM, PBS, Amazon AWS, ...).
21

22. Beware of Pipelineitis
• “Pipelines” can kill your creativity and force
you to think too rigidly.
• Don’t “pipeline” too early, if at all.
• Does it even need to be pipeline-ified?
• Who’s running it?
- You, once: don’t pipeline-ify. Document, move along.
- You, 2-5 times: documented script?
- You, 10+ times: consider pipeline-ifying.
- Others: create sharable pipeline
• See: Loman & Watson. "So you want to be a
computational biologist?" Nat Biotechnol 31
(2013): 996-998.
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23. Documentation
23

24. Dynamic Documentation: RMarkdown
• R: widely used for data science & bioinformatics
• Markdown: a simple markup language that allows you to
render structured/formatted documents from plain text.
• RMarkdown: embeds R code in a Markdown
document.
- Write documents that execute embedded code and
integrates results into the final report.
- Allows you to keep code and documentation together.
- Easily re-render the document, re-running analysis
and re-incorporating results on the fly.
- Many output formats: PDF, DOCX, HTML, EPUB, ...
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25. 25
Write plain text document
Embed R code
Rendered output report

26. 26
output: pdf_document output: word_document

27. Jupyter notebooks
• jupyter.org
• Jupyter: open source project to
develop software, standards,
services across many languages
• Jupyter notebook: free
application to create documents
containing live code,
visualizations, narrative text.
- Supports >40 programming
languages
- Easily shared
- Interactive output
- Multi-user versions for
companies, classrooms, labs
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28. Data / code sharing
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29. Sharing Code
• State of the art early-2000's
- "Data/code available upon request"
- "Code available on <lab website>"
- None of the above
• Schultheiss, Sebastian J., et al. "Persistence and
availability of web services in computational
biology." PloS one 6.9 (2011): e24914.
- Surveyed ~1000 web services published in NAR
2003-2009
- ~30% unavailable
- ~80% developed by students / non-permanent
researcher
• Russell, Pamela H., et al. "A large-scale analysis of
bioinformatics code on GitHub." bioRxiv (2018):
321919.
• github.com is becoming the de facto standard for
archiving and sharing code
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30. Sharing any research output
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- figshare.com
- Free
- Upload any file format
- Get a DOI
- 5 GB max file size
- 20GB private space
- Unlimited public space
- Launched 2012
- Hosted on S3, multiple
redundant copies
- SLA: 10 yr persistence
- zenodo.org
- Free
- Upload any file format
- Get a DOI
- 50 GB per record
- Higher quota by request
- Unlimited records
- Launched 2013
- Hosted at CERN (est
1954), with defined
program of ≥20 years
- about.zenodo.org/policies/
- about.zenodo.org/principles/
- osf.io
- Free
- Upload any file format
- Get a DOI
- 5 GB per file
- Connect to any external
storage provider
- Launched 2013
- Preservation fund
guaranteeing 50+ years
of persistent availability
- osf.io/faq

31. 31
doi.org/10.5281/zenodo.1255003
bit.ly/madssci2018repro
Slides:

32. Other Resources
32
Wilson, et al. "Good enough practices
in scientific computing." PLoS
computational biology 13.6 (2017):
e1005510.
Wilson, et al. "Best practices for
scientific computing." PLoS
biology 12.1 (2014): e1001745.
https://doi.org/10.1371/journal.pbio.1001745
https://doi.org/10.1371/journal.pcbi.1005510

33. Other Resources
• 2017: Ten simple rules for making research software more robust:
http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1005412
• 2017: Ten simple rules for responsible big data research:
http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1005399
• 2017: Ten Simple Rules to Enable Multi-site Collaborations through Data Sharing:
http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1005278
• 2016: Ten Simple Rules for Digital Data Storage:
http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1005097
• 2016: Ten Simple Rules for Effective Statistical Practice:
http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1004961
• 2015: Ten Simple Rules for Creating a Good Data Management Plan:
http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1004525
• 2015: Ten Simple Rules for Experiments’ Provenance:
http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1004384
• 2015: Ten Simple Rules for a Computational Biologist’s Laboratory Notebook:
http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1004385
• 2015: Ten Simple Rules for Reducing Overoptimistic Reporting in Methodological Computational Research:
http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1004191
• 2014: Ten Simple Rules for the Care and Feeding of Scientific Data:
http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1003542
• 2014: Ten Simple Rules for Effective Computational Research:
http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1003506
• 2013: Ten Simple Rules for Reproducible Computational Research:
http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1003285
• 2012: Ten Simple Rules for the Open Development of Scientific Software:
http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1002802
• 2014: Ten Simple Rules for Writing a PLOS Ten Simple Rules Article:
http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1003858
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collections.plos.org/
ten-simple-rules

34. Other Resources
• Baker, Monya. “1,500 Scientists Lift the Lid on Reproducibility.” Nature News, vol.
533, no. 7604, May 2016, p. 452. www.nature.com, doi:10.1038/533452a.
• Grüning, Björn, et al. “Practical Computational Reproducibility in the Life Sciences.”
BioRxiv, Oct. 2017, p. 200683. www.biorxiv.org, doi:10.1101/200683.
• Leek, Jeff. "A Few Things That Would Reduce Stress around Reproducibility/
Replicability in Science." Simply Statistics, November 2017: https://simplystatistics.org/
2017/11/21/rr-sress/.
• Mesirov, Jill P. “Accessible Reproducible Research.” Science, vol. 327, no. 5964, Jan.
2010, pp. 415–16. science.sciencemag.org, doi:10.1126/science.1179653.
• Munafò, Marcus R., et al. “A Manifesto for Reproducible Science.” Nature Human
Behaviour, vol. 1, no. 1, Jan. 2017, p. 0021. www.nature.com, doi:10.1038/
s41562-016-0021.
• Patil, Prasad, et al. “A Statistical Definition for Reproducibility and Replicability.”
BioRxiv, July 2016, p. 066803. www.biorxiv.org, doi:10.1101/066803.
• Russell, Pamela, et al. “A Large-Scale Analysis of Bioinformatics Code on GitHub.”
BioRxiv, May 2018, p. 321919. www.biorxiv.org, doi:10.1101/321919.
• Schultheiss, Sebastian J., et al. “Persistence and Availability of Web Services in
Computational Biology.” PLOS ONE, vol. 6, no. 9, Sept. 2011, p. e24914. PLoS
Journals, doi:10.1371/journal.pone.0024914.
34

35. Stephen D. Turner, Ph.D.
Bioinformatics Core Director
University of Virginia School of Medicine
bioinformatics.virginia.edu
@strnr
THANKYOU
bit.ly/madssci2018repro
doi.org/10.5281/zenodo.1255003